Two approaches have been used by this laboratory for the isolation of new oncogenes: (1) transduction with retrovirus and (2) transformation of cells by retrovirus insertion followed by molecular cloning of flanking DNA. The first approach has yielded the v-raf oncogene-carrying virus 3611MSV and several other isolates which will be molecularly analyzed in the future. For the second approach an in vitro system was established for the transformation of rodent epithelial cells by C3H MuLV in conjunction with 12-0-tetradecanoylphorbol-13-acetate (TPA). Transformed cell clones obtained from soft agar generally were virus nonproducers. The purpose of these experiments was to sequence-label TPA-promotable cellular tumor genes and, thus, make possible their isolation by molecular cloning. Epithelial rodent cells that had been transformed with MuLV and TPA are being examined for expression of novel virus-cell hybrid transcripts and are being used for the molecular cloning of LTR-linked cellular DNA. As an example for the principle effectiveness of such a strategy, we have demonstrated for NIH 3T3 cells transformed by transfection with MuLV LTR DNA that the proto-oncogene, c-raf-1, was activated as an oncogene by a promoter insertion mechanism. Moreover, we have recently described, for in vitro-infected and transformed myeloid cells, integration of MuLV into the sixth exon of the myb proto-oncogene. The effect of integration was a truncation of the myb protein which may contribute to its transforming ability. Integration was precisely at the site previously identified for insertional activation of c-myb in vivo by another strain of MuLV.